Anti-m lectin from seeds of cruciferae iberis



. 3,035,986 ANTIi-M LECTW FROM SEEDS F CRUCIFERAE IBERHS Roy T. Fisk, Glendale, and Nancy K. Allen, Les Angeles, Galifi, assignors to Hyland Laboratories, Los

Angeles, Calif., a corporation of California No Drawing. Filed Ian. 2, 1959, Ser. No. 784,455

Claims. (Cl. 167-84.5)

This invention relates to compositions which are useful reagents for the diagnosis and characterization of blood.

Landsteiner discovered that human blood shows individual differences characterized by agglu-tinogens carried by erythrocytes and by agglutinins present in blood serum. The agglutinogens which comprise the four major blood groups were identified by means of anti-A and anti-B agglutinins which are of natural or normal occurrence in serums of individuals not possessing the corresponding agglutinogen. Group 0 persons lack both A and B cellular agglutinogens and develop both anti-A and anti-B. agglutinins early in life. Group A individuals, however, inheret the A agglutino-gen and develop agglutinins only to the B agglutinogen which they lack, while group B persons form only anti-A agglutinin. The rarer AB group develops neither of these agglutinins.

Group B serum is a ready source of anti-A agglutinins and group A serum affords an anti-B grouping reagent. The natural occurrence of these agglutinins was Landsteiners key for unlocking the mystery of blood incompatibility which up to that time made transfusion of this important agent hazardous, if not impossible. The subsequent identification of other important blood agglutinogens by Landsteiner and by others have made whole blood transfusion an even safer procedure. In this connection, and listed in the order of their historical identification, are the more important of these agglutinogens: M, N, P, Rh and Hr.

It should be noted that the discovery of these agglutinogens was much more difiicult than those of the major blood groups because of the lack of naturally occurring agglutinins to them in human blood. Specific agglutinins necessary for identifying certain agglutinogens (M, N. P, and Rh) were obtained by immunizing laboratory animals. Some agglutinogens (H (O) and P) were identified by analyzing the normal serums of various animals or by studying agglutinins developed by individuals who had become immunized or sensitized to a particular blood type agglutinogen. Agglutinins from sensitized human beings are at present the only practical source of anti-Rh agglutinins (anti-C, anti-D, anti-E, anti-c, and anti-e).

The understanding of blood groups and blood types has enlarged considerably since the discovery of the Rh factor by Landsteiner and Wiener in 1940. Recent observations contribute to a growing list of blood cell agglutinogens, as well as their corresponding agglutinins, and methods used in detecting them are of interest to those in allied fields of study. These findings are of particular significance in medicine and laboratory diagnosis, as for example, in ascertaining blood compatibility prior to transfusion and in the field of legal medicine where the presence or absence of blood agglutinogens serves as evidence in disproving parentage. Also, blood group and type relationships worked out by geneticists have given anthropologists a valuable instrument for racial studies.

Although blood has been the principal source or" agglutinins for use in blood typing, it is not the only source. It has long been known that products of certain bacteria, a number of organic and inorganic chemical substances, and extracts of the seeds of certain plants will cause the agglutination of red blood cells. None of these, however, were of use in blood typing because they were non specific. That is, if they agglutinated the red cells of one individual, they agglutinated the red cells of all other human beings, and consequently did not divide them into groups. In 1945 Dr. W. C. Boyd discovered that extracts of some seeds were specific for certain blood group antigens (i.e., they agglutinated only blood of certain blood groups), and thereby established the possibility of using such extracts in blood grouping. Such a seed extract reacts specifically with a blood cell factor and behaves very muchlike a specific agglutinin of human or animal origin. It is believed that this specificity is of an accidental nature and that the plant forms the agglutinin-like substance without agglutinogenic stimulation. For this reason, Dr. Boyd proposed the term lectins for the blood-group-specific plant agglutinins, in order to avoid the use of the word antibody which would imply that they were the result of immunization, which may or may not be the case.

Lectins of anti-A, anti-A (a sub-group of type A), anti-B, anti-N and anti-O specificity are known to exist; and, of these, anti-A and anti-H(O) lectins have been made commercially available. There is however, no commercially available anti-M specific leotin. A preparation of thistype is of considerable practical importance in View of the well-known difilculty of making good anti-M agglutinin from immune rabbit serum.

It is an object of this invention to provide a new source of anti-M lectin. Another object is to provide an anti-M lectin which is useful in the formulation of diagnostic compositions which have commercial significance. Another object is to provide an anti-M lectin which is characterized by a high degree of sensitivity and specificity. Another object is to provide novel procedures for the isolation and preparation of anti-M lectins. Other objects and advantages, both general and specific, will be apparent from the following detailed description of the invention.

This invention relates to the preparation and use of lectins, derived from the seeds of Cruciferae Iberis, as diagnostic reagents for the identification of human erythrocytes of types M and MN.

In the products employed in the practice of this invention, the seeds of Cruciferae Iberis, a plant more commonly known as common candytuft, variety Iceberg, are extracted by procedures described in greater detail hereinafter. Anti-M lectins suitable for routine use as anti-M diagnostic reagents are obtained by purification and conceneration of these extracts.

The Cruciferae Iberis lectin of this invention is prepared by reducing the seeds of the plant to a small, particulate form, such as by mechanical attrition. The lectin is solubilized at reduced temperatures, for example, 2 to 10 degrees (3., through the use of a suitable solvent, such as an aqueous solution of sodium chloride; and any insoluble inert material is discarded. The extract may be clarified by centrifugation and filtration through a medium which neither adsorbs nor adversely affects the active principal. The extract is then further purified by dialysis. The leotin is found in the precipitate which results from the dialysis. This precipitate is solubilized through the use of a suitable solvent such as an aqueous 20% solution of sodium chloride, and the insoluble inert material is removed and discarded.

It has been observed that the activity of the purified anti-M lectin prepared from Cruciferae Iberis, according to the methods described herein, can be significantly increased in the presence of acacia. Acacia itself has no demonstrable anti-M activity. It is apparent, therefore, that the anti-M lectin of this invention and acacia constitute a synergistic combination. Compositions which incorporate this synergistic combination are preferred for use in the practice of this invention.

The following detailed example will serve to more fully describe the invention.

Example I An anti-M lectin was obtained from Cruciferae Iberis seeds in the following manner. The seed was finely ground by mechanical means and then extracted in ten times its weight of 0.9% sodium chloride solution. The extraction was continued at room temperature for 8 hours, with occasional mixing, and was continued overnight at a temperature of 4 degrees C. The extraction was clarified by centrifugation and filtration through glass wool. This crude extract, which contained the active principal, was then dialyzed against distilled water for 24 hours in 12 changes of water. Each time the water was changed, the extract was thoroughly mixed by inverting the Visking tubing several times. The insoluble ma terial which resulted from this dialysis was collected by centrifugation, and was solubilized by the addition of one-fourth volume of sodium chloride solution.

This 20% sodium chloride solution of the precipitate was then diluted with a suflicient quantity of 0.9% sodium chloride solution to provide a volume equal to one-half the volume of the original crude extract, which was subjected to dialysis. This solution was then tested for anti-M activity, using the following technique:

(1) Place two drops of the lectin preparation in each of three suitable, e.g., 10 x 75 mm., test tubes.

(2) Add to one of these test tubes one drop of a 2% by volume suspension of erythrocytes of M specificity, and 0.9% sodium chloride solution, and mix. Repeat this procedure for each of the other two test tubes, using erythrocytes of MN and N specificity, respectively.

(3) Prepare negative controls, each with two drops of sodium chloride solution and one drop of erythrocytes of M, MN, and N specificity, respectively.

(4) Allow the test tubes to remain at room temperature for one hour.

(5) Resuspend the sediment which forms in the tubes and centrifuge for one minute at 1700 r.p.m.

(6) Shake the tubes gently and observe for macroscopic agglutination.

(7) Extracts which contain lectin specific to the erythrocyte agglutinogens produce visible floculation of the erythrocytes, whereas a smooth suspension is observed in a negative reaction.

When the above test procedure was employed, the lectin preparation of this example caused a visible agglutination of the cells which were known to possess M and MN agglutinogens. This lectin preparation did not cause a visible agglutination of the cells which were known to possess vonly the N agglutinogen. These results demonstrate the presence of an M specific lectin in the seeds of Cruciferae Iberis. They further demonstrate that this lectin can be extracted from these seeds and purified in a manner that will provide a commercially acceptable lectin preparation.

Example I1 One volume of the purified lectin preparation of Example I was admixed with two volumes of an aqueous 3% acacia solution. This admixture was tested for lectin activity according to the procedures set forth in Example I. It was observed that the admixture retained the M specificity of the original lectin preparation and provided stronger and more clear-cut agglutination reactions than were provided by the lectin preparation which did not contain acacia. Furthermore, although a one hour incubation period was required for the lectin preparation of Example I, the improved results obtainable with the admixture employed in this example, could be realized in 15 minutes. The 3% acacia solution itself, that is, without the added presence of lectin, displayed no agglutinating properties.

While in the foregoing specification, a detailed description of embodiments of the invention has been set forth for the purpose of illustration, it will be apparent to those skilled in the art that many modifications in the details of these embodiments may be made without departing from the spirit and principles of the invention.

There is claimed:

1. In a method of preparing an agglutinin which is specific for the M agglutinogen of blood, the step of particulating seeds of Cruciferae Iberis, mixing the particulated seeds with an aqueous solvent to dissolve the agglutinin portions thereof, discarding the insoluble portions of the particulated seeds, removing inactive material from the said agglutinin portions by dialysis, and combining the residual agglutinin with acacia.

2. In a method of preparing an agglutinin which is specific for. the M agglutinogen of blood, the step of par-' ticulating seeds of Cruciferae Iberis, mixing the particulated seeds with an aqueous solvent to dissolve the agglutinin portions thereof, discarding the insoluble portions of the particulated seeds, and removing the inactive material from the seed agglutinin portions by dialysis.

3. In a method of preparing an agglutinin which is specific for the M agglutinogen of blood, the steps of particulating seeds of Cruciferae Iberis, mixing the particulated seeds with an aqueous electrolyte solvent to dissolve the agglutinin portions thereof, discarding that portion of the particulated seeds, which is insoluble in the aqueous electrolyte solvent, removing dialyzable, inactive material from the agglutinin portions by dialysis, and thereafter recovering the non-dialyzable water-insoluble agglutinin.

4. In a method of preparing an agglutinin which is specific for the M agglutinogen 0 blood, the steps of particulating seeds of Cruciferae I eris mixing the particulated seeds with an aqueous electrolyte solvent to dissolve the agglutinin portions thereof; discarding that portion of the particulated seeds which is insoluble in the aqueous electrolyte solvent; removing dialyzable, inactive material from the agglutinin portions by dialysis; recovering the non-dialyzable, water-insoluble agglutinin; and combining the agglutinin with acacia.

5. The agglutinin-acacia product resulting from the process of claim 1.

References Cited in the file of this patent UNITED STATES PATENTS Eldon Nov. 13, 1956 OTHER REFERENCES 

1. IN A METHOD OF PREPARING AN AGGLUTININ WHICH IS SPECIFIC FOR THE M AGGLUTINOGEN OF BLOOD, THE STEP OF PARTICULATING SEEDS OF CRUCIFERAE IBERIS, MIXING THE PARTICULATED SEEDS WITH AN AQUEOUS SOLVENT TO DISSOLVE THE AGGLUTININ PORTIONS THEREOF, DISCARDING THE INSOLUBLE PORTIONS OF THE PARTICULATED SEEDS, REMOVING INACTIVE MATERIAL FROM THE SAID AGGLUTININ PORTIONS BY DIALYSIS, AND COMBINING THE RESIDUAL AGGLUTININ WITH ACACIA. 